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J Bacteriol. 2006 October; 188(20): 7267–7273.
PMCID: PMC1636243

Blocking Chromosome Translocation during Sporulation of Bacillus subtilis Can Result in Prespore-Specific Activation of σG That Is Independent of σE and of Engulfment

Abstract

Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase σ factors, which are activated in the order σFEGK. Of these, σG becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding σG, spoIIIG, is directed in the prespore by RNA polymerase containing σF but also requires the activity of σE in the mother cell. When first formed, σG is not active. Its activation requires expression of additional σE-directed genes, including the genes required for completion of engulfment. Here we report conditions in which σG becomes active in the prespore in the absence of σE activity and of completion of engulfment. The conditions are (i) having an spoIIIE mutation, so that only the origin-proximal 30% of the chromosome is translocated into the prespore, and (ii) placing spoIIIG in an origin-proximal location on the chromosome. The main function of the σE-directed regulation appears to be to coordinate σG activation with the completion of engulfment, not to control the level of σG activity. It seems plausible that the role of σE in σG activation is to reverse some inhibitory signal (or signals) in the engulfed prespore, a signal that is not present in the spoIIIE mutant background. It is not clear what the direct activator of σG in the prespore is. Competition for core RNA polymerase between σF and σG is unlikely to be of major importance.

Formation of spores by Bacillus subtilis is a simple two-cell differentiation process that has become a paradigm for studying cell differentiation in prokaryotes. Central to this process is compartmentalization of gene expression between the two cell types. Sporulation involves asymmetrical division that yields two cells of different sizes, which have different developmental fates. The smaller cell, called the prespore (or forespore), ultimately develops into the mature, heat-resistant spore. The larger cell, called the mother cell, is necessary for spore formation but ultimately lyses. Expression of different genes in the two cells is governed by the activation of four RNA polymerase sigma factors: σF and then σG in the prespore and σE and then σK in the mother cell (for a review, see reference 14). Each of these sigma factors directs expression of distinct regulons, which have recently been delineated by microarray analysis; the smallest regulon, the regulon for σF, contains about 50 genes, and the largest, the regulon for σE, contains perhaps 200 genes (7, 42, 49).

The first sigma factor to become active, σF, does so soon after formation of the spore septum, and its activation leads rapidly to the activation of σE (12; for a review, see reference 14). Development continues with the mother cell engulfing the prespore. Upon completion of engulfment, the prespore is entirely within the mother cell and so is no longer in direct contact with the medium. At this time, σG becomes active in the prespore, and it in turn triggers activation of σK in the mother cell. Coordination of the cascade of sigma factor activation is ensured by intracellular controls and by intercellular communication between the two compartments (14, 25).

In this report, we focus on the activation of σG, which is encoded by the spoIIIG locus (27, 47). The spoIIIG locus is first transcribed during sporulation by read-through from the spoIIG locus. However, the transcript is translated poorly, if at all, into σG (27, 45); it is not needed for spore formation (45), and its role is obscure. Productive transcription of spoIIIG is initiated from a promoter immediately upstream of the gene. It is directed by RNA polymerase containing σF and so is confined to the prespore (17). This transcription is delayed compared to that of other σF-directed genes (19, 42), and it requires a σE-directed signal from the mother cell (17, 29). It also requires expression of the σF-directed spoIIQ locus (48); since the SpoIIQ protein is located in the membrane, its regulation of spoIIIG transcription is also thought to be indirect (34). Placing spoIIIG under a strong σF-directed promoter (44) or mutating the spoIIIG promoter (10) can result in σE-independent transcription of spoIIIG. However, this does not make activation of σG independent of σE, indicating that there are additional controls dependent on σE; indeed, these controls appear to be more important for spore formation (10).

When first formed, σG is not active. Its activation depends on completion of engulfment of the prespore (43), which requires the activity of several σE-directed genes (1, 14). Activation also requires the σE-directed expression of the spoIIIA operon, whose products are thought to transmit some signal from the mother cell to the prespore via the SpoIIIJ protein (8, 38). It is not clear if the SpoIIIA-mediated signal is distinct from the signal that engulfment is completed, as strains with spoIIIA mutations complete engulfment (30). A protein encoded in the spoIIIA operon, SpoIIIAH, interacts with the prespore protein SpoIIQ, suggesting that there is an additional regulatory mechanism that acts via SpoIIIA (2). However, it remains to be established whether SpoIIQ does indeed have a role in σG activation in addition to its role in spoIIIG transcription (2, 48).

There are additional controls that prevent σG from becoming active in the other cell type, the mother cell (5, 40). These controls have complicated analysis of the controls of prespore activation. For example, mutations that bypassed the need for spoIIIA in σG activation resulted in deregulation of σG expression in the mother cell but not in the prespore (5, 40). Such mutations overcame SpoIIAB-mediated inhibition of σG in the mother cell, a regulatory system that may be quite separate from the SpoIIIA-SpoIIIJ system regulating σG in the prespore.

Here we focused on σG activation in the prespore. We found that it is possible to activate σG in the prespore independent of σE activity and of spoIIQ; in the conditions used, σG became active after septum formation and before engulfment was initiated. These results suggest that σE/SpoIIQ regulation normally ensures that σG does not become active until engulfment is completed. Premature σG activation does not curtail σF activity, suggesting that competition for core polymerase is not a major factor in determining σG activation or σF inactivation.

MATERIALS AND METHODS

Media.

B. subtilis was grown in modified Schaeffer's sporulation medium (MSSM) or on Schaeffer's sporulation agar (31, 35). When required, the medium contained 5 μg chloramphenicol/ml, 1.5 μg erythromycin/ml, 3.5 μg neomycin/ml, 100 μg spectinomycin/ml, or 10 μg tetracycline/ml. Escherichia coli was grown on Luria-Bertani lysogeny broth agar containing 100 μg ampicillin/ml when required.

Strains.

B. subtilis 168 strain BR151 (trpC2 metB10 lys-3) was used as the parent strain. The B. subtilis strains used are listed in Table Table1.1. Strain PS1120 with spoIIIG inserted at amyE (45) was kindly provided by Peter Setlow (Connecticut Health Sciences Center); the amyE::spoIIIG construct was introduced by transformation into strains listed in Table Table1,1, with selection for the linked cat marker. The spoIIIE36 mutation is spo-36 described by Hranueli et al. (15). Mutants with spoIIR inactivated were constructed by double crossover at the spoIIR locus in such a way that 342 bp at the 3′ end of the gene was replaced with PsspA-lacZ or PsspA-gfp transcriptional fusions linked to an antibiotic resistance cassette. The fusions were designed so that their expression could not be driven by the spoIIR promoter or by the promoter for the resistance cassette. The PspoIIR-cfp and PspoIIR-lacZ fusions were inserted at the spoIIR locus by double crossover, disrupting the locus. The PsspA-yfp fusion was present in a derivative of the pAMβ1 replicon shuttle plasmid pJAR2 (3). Vectors with the gfp, cfp, and yfp genes were kindly provided by W. G. Miller (28), D. Z. Rudner (6), and P. J. Lewis (11), respectively. E. coli DH5α (Gibco-BRL) was used to maintain plasmids. Details of plasmid and strain construction are available on request.

TABLE 1.
B. subtilis strains used

Fluorescence microscopy.

Cultures were grown in MSSM at 37°C. A 200-μl portion of culture was mixed with 0.2 μl of a 1-mg/ml stock solution of FM4-64 (Molecular Probes) in phosphate-buffered saline (Gibco-BRL). Samples were incubated at 37°C for 5 min, and 1 μl of an unfixed sample was transferred to a slide and visualized essentially as described by Pogliano et al. (33). Images were captured using a Leica DM IRE2 microscope with a TCS SL confocal system as described previously (5). Excitation for green fluorescent protein (GFP) and FM4-64 was at 488 nm; emission for GFP was captured at wavelengths between 500 and 550 nm, and emission for FM4-64 was captured at wavelengths between 600 and 730 nm. Excitation for cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) was at 458 and 514 nm, respectively, and emission was captured at wavelengths between 465 and 500 and between 525 and 550 nm, respectively. In general, there was 4× line averaging and 3× frame averaging.

Other methods.

β-Galactosidase activity was assayed essentially as described previously (3). Specific activity was expressed in nanomoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per milligram (dry weight) of bacteria. Results of typical experiments are shown below; the activities are the means for duplicate cultures in an experiment. The methods used for transformation of B. subtilis and for sporulation by exhaustion in MSSM and other methods were essentially the methods described previously (31).

RESULTS

Rendering σG activation in the prespore independent of σE activity.

Productive transcription of spoIIIG, the structural gene for σG, is initiated from a σF-directed promoter and takes place in the prespore (45). This σF-directed transcription normally requires activation of σE in the mother cell (17, 29). When the spoIIIG promoter was relocated to the origin-proximal amyE locus, its transcription, as assayed with a PspoIIIG-lacZ transcriptional fusion, still required σE activity (10, 17) (Fig. (Fig.1,1, compare strains SL5430 and SL10117). However, we found that when translocation of the origin-distal 70% of the chromosome into the prespore was blocked by a spoIIIE36 mutation, σE activity was no longer required for the σF-directed PspoIIIG-lacZ transcription (Fig. (Fig.1,1, compare strains SL10086 and SL11106); increased σF-directed transcription in a spoIIIE mutant background has been observed previously for σF-directed genes (37, 39, 46). The transcription of spoIIIG remained absolutely dependent on σF (data not shown).

FIG. 1.
The PspoIIIG promoter is expressed in the absence of σE when it is located at the origin-proximal amyE locus in a spoIIIE36 mutant. Transcription directed by the PspoIIIG promoter was assessed by determining the β-galactosidase activity ...

Activity of σE is normally required for the activation of σG, as well as for the transcription of spoIIIG. The observations described above for the transcription of spoIIIG encouraged us to test whether σG became active in the absence of σE when chromosome translocation was blocked by a spoIIIE36 mutation. To monitor σG activity, a PsspA-lacZ transcriptional fusion was introduced into the origin-proximal spoIIR locus. This insertion inactivated spoIIR and so prevented activation of σE (18); loss of σE activity in strains with this construct was confirmed by their failure to initiate engulfment and also by the absence of expression of a σE-dependent PspoIID-lacZ fusion when it was introduced by appropriate crosses (data not shown). The spoIIIE36 mutation prevented translocation of the origin-distal spoIIIG locus into the prespore and so prevented its σF-directed transcription (52). Consistent with this result, no σG activity was detected in a spoIIIE36 mutant, strain SL12918, in which spoIIIG is at its natural locus (Fig. (Fig.2).2). However, when the spoIIIG locus was inserted into the origin-proximal amyE locus, σG activity was readily detected (Fig. (Fig.2,2, strain SL12916). The level of σE-independent σG activity in strain SL12916 was similar to the level of σG activity of a spo+ strain, SL10369 (Fig. (Fig.2),2), although expression appeared to start slightly earlier, during spore formation. Thus, σE activity was not required for either transcription of spoIIIG or σG activation. However, there was still a requirement for σF activity, as PsspA-lacZ expression was blocked by inactivation of spoIIAC, the structural gene for σF (Fig. (Fig.2,2, strain SL12938).

FIG. 2.
Activation of σG independent of σE in a spoIIIE36 mutant with spoIIIG located at amyE. The activity of σG was assessed by determining the β-galactosidase activity in the following strains: SL10369 (spo+ PsspA-lacZ@sspA ...

σE-independent activation of σG is confined to the prespore.

To test the location of the σE-independent activation of σG in an amyE::spoIIIG spoIIIE36 strain, SL12864 was constructed, in which a σG-dependent PsspA-gfp transcriptional fusion was inserted into the spoIIR locus. Because of the absence of σE activity, this strain underwent sporulation division but did not initiate engulfment. Strain SL12864 expressed GFP, and expression was confined to the prespore (Fig. (Fig.3B3B and Table Table2);2); similar results were obtained when spoIIGB, which is the structural gene for σE, was also inactivated (data not shown). Mutants lacking σE activity often form prespores at both ends of the sporulating organism (the abortively disporic phenotype [16, 30]). This phenotype was observed with SL12864, and in the majority of the disporic cells the GFP signal was detected in both prespores (Fig. (Fig.3B).3B). The results confirmed that the σE-independent σG activity is confined to the prespore. In the spo+ strain, SL10969, the prespore-specific σG activity was detected only in bacteria that had completed engulfment (Fig. (Fig.3A),3A), whereas in SL12864 it was detected at the preengulfment stage (Fig. (Fig.3B3B).

FIG. 3.
Location of σE-independent σG activity. (A to C) Examples of bacteria stained with FM4-64 (red) and expressing GFP (green) under control of the σG-directed sspA promoter. (A) SL10969 (spo+); (B) SL12864 (spoIIIE36 amyE ...
TABLE 2.
Location of fluorescent protein expressed from σG- and σF-directed promotersa

The efficiency of PsspA-gfp expression in SL12864 was comparable to that in a spo+ strain (SL10969) in which the PsspA-gfp fusion was inserted at sspA and to that of the spoIIIE36 strain SL12929, in which the activity of σE was restored by complementing spoIIR (Table (Table2).2). These data are in agreement with the quantitative analysis of PsspA-lacZ expression in the two genetic backgrounds, as mentioned above. In spo+ strains, σG does not become active in the prespore until after completion of engulfment (32), whereas the σE-independent activation of σG occurs after the sporulation division and before initiation of engulfment (indeed, engulfment is not completed in strains that lack σE activity). The mechanism of σE-dependent activation of σG includes, but is probably not limited to, expression of the spoIIIA locus (8, 38). Whatever the details, this mechanism is not required when the origin-distal 70% of the chromosome is trapped in the mother cell and spoIIIG is located in the prespore.

In order to simultaneously visualize both σF and σG activities, we constructed strain SL12972 with PspoIIR-cfpF-directed) and PsspA-yfpG-directed) transcriptional fusions. The PspoIIR-cfp fusion was inserted into and disrupted spoIIR. The PsspA-yfp fusion was present in an autonomously replicating plasmid; increasing the gene dosage of sspA had been shown previously to cause only a modest increase in its mRNA level (26). The strain containing the fusions also had a spoIIIE36 mutation and spoIIIG located at amyE. The activities of both σF and σG were confined to the prespores (Table (Table2,2, strain SL12972, and Fig. 3D to I). The two activities were generally detected in the same preengulfment prespores (Table (Table2)2) in both monosporic and disporic organisms.

Restoration of σE activity to a spoIIIE36 amyE::spoIIIG mutant does not change the timing of σG activation.

During the normal course of spore formation, σG does not become active until after completion of engulfment, and its activation depends on the activity of σE. Above, we describe conditions in which σG became active in the preengulfment prespore and in the absence of σE. We were interested to see if restoration of σE activity in these conditions affected the timing or location of σG expression. In the strains used, σE was not active because of the loss of spoIIR. In order to restore σE activity, a functional copy of spoIIR was introduced into strain SL12864 by single (Campbell-like) crossover at the truncated spoIIR locus, yielding strain SL12929. The restoration of spoIIR indeed resulted in activation of σE, as indicated by bacteria proceeding toward the completion of engulfment and by the expression of a σE-dependent PspoIID-lacZ fusion when it was introduced into the strain (data not shown). In strain SL12929, σG activity was confined predominantly to the prespore. Importantly, it appeared after completion of the sporulation division septum and before the initiation of engulfment was discernible. The proportion of cells in which σG activity was detected for strain SL12929 was similar to the proportion for strain SL12864, in which σE was not active (Table (Table22 and Fig. Fig.3C).3C). In strain SL12929, substantial numbers of bacteria proceeded toward the completion of engulfment (Fig. (Fig.3C),3C), consistent with the restoration of σE activity in a spoIIIE36 mutant. The σG activity in SL12929 was initially prespore specific, although during prolonged incubation there was a breakdown of compartmentalization accompanied by extensive cell lysis, as is typical of spoIIIE mutants in which the prespores are unstable and lyse (data not shown) (4, 22, 43). The effect of restoration of σE activity on the time of σG expression was also tested by integrating a functional copy of spoIIR into strain SL12916 by single crossover. The expression of the σG-directed sspA-lacZ fusion in the resulting strain, SL12936, was very similar to the expression in strain SL12916, in which σE was not active (Fig. (Fig.2).2). Restoration of σE activity did not restore σK activity to the spoIIIE36 mutant strains (data not shown).

Activation of σG in the prespore does not markedly curtail σF activity.

Sigma factors σF and σG are very similar to each other (13). They have overlapping promoter specificities (13), and both are inhibited by the anti-sigma factor SpoIIAB (9, 20, 40). During normal spore formation, they are activated successively in the prespore, and there are indications that σF activity is curtailed when σG becomes active (23). Thus, σF and σG may be in direct competition with each other. One possibility is that σG outcompetes σF for core RNA polymerase and supplants it. Alternatively, σF might outcompete σG and prevent it from becoming active until some other factor removes σF from the competition. The σE-independent activation of σG described above provided the possibility to test the effect of direct competition between σF and σG in the preengulfment prespore.

There was little difference in σF activity between strains SL12857 and SL12861 (Fig. (Fig.4),4), which differ only in the presence of an expressed copy of spoIIIG located at amyE in strain SL12857. Thus, the activity of σG in the preengulfment prespore does not seem to have an appreciable effect on σF activity. The strains contained a PspoIIR-lacZ fusion inserted at spoIIR to assay σF activity; the insertion disrupted spoIIR and so prevented σE activation.

FIG. 4.
Effect of σG activity on σF activity in the preengulfment prespore. The activity of σF was assessed by determining the β-galactosidase activity in the following strains: SL12857 (spoIIIE36 spoIIR::PspoIIR-lacZ) (○) ...

This result reinforces the conclusion from studies of a lonA mutant that competition between σF and σG for core polymerase may not be important in determining the activities of these sigma factors (23). We infer that competition between the sigma factors for core RNA polymerase does not explain the normal delay in activation of σG until after the completion of engulfment that occurs in spo+ strains.

Expression of σG activity in an amyE::spoIIIG spoIIIE36 mutant is independent of spoIIQ.

The appearance of σG activity ordinarily requires expression of the σF-directed spoIIQ locus (48). This expression is prespore specific (24) and so might regulate σG expression separately from the σE-directed mother cell signal(s). Transcription of spoIIIG directed by σF normally depends on expression of the spoIIQ locus, even when spoIIIG is relocated to amyE (47); it is possible that the SpoIIQ protein is required for activation of σG, as well as for spoIIIG transcription (2). We wanted to test whether this dependence on spoIIQ was retained in a spoIIIE36 amyE::spoIIIG strain. For this purpose, the spoIIQ mutant SL13225 was constructed. Strain SL13225 displayed β-galactosidase activity in sporulation conditions (Fig. (Fig.5)5) similar to that of the isogenic spoIIQ+ strain, SL12916 (Fig. (Fig.5),5), except that at later times (6 to 7 h after the end of exponential growth) strain SL13225 displayed somewhat more activity than the spoIIQ+ strain. Thus, inactivation of spoIIQ did not impair σG activity in a spoIIIE36 amyE::spoIIIG strain, indicating that spoIIIG transcription and σG activation did not require either SpoIIQ or σE in this genetic background. Indeed, the only effect of spoIIQ inactivation was to increase σG activity at later times.

FIG. 5.
Activation of σG is independent of SpoIIQ in a spoIIIE36 amyE::spoIIIG background. The activity of σG was assessed by determining the β-galactosidase activity in the following strains: SL12916 (spoIIIE36 amyE::spoIIIG spoIIR::P ...

DISCUSSION

Gene expression during formation of spores by B. subtilis is controlled by the successive activation of RNA polymerase sigma factors in the order σFEGK through a complex pattern of intra- and intercellular signals, in which activation of the later sigma factors in the sequence depends on activation of the earlier factors (25, 32). Thus, the activation of σG in the prespore, which occurs upon completion of engulfment, depends on prior activation of σF in the prespore and prior activation of σE in the mother cell. We report here conditions in which σG is activated in the prespore before engulfment is completed and independent of σE activity. To our knowledge, this is the first report of overlapping σF and σG activities in the preengulfment prespore. The premature prespore-specific σG activity depends on the genetic background of the strains used. In the strains used the origin-distal 70% of the chromosome was retained in the mother cell, and spoIIIG, the structural gene for σG, was inserted into an origin-proximal site, which was present in the prespore. The relocation of spoIIIG is necessary for its transcription to be directed by σF, which is active only in the prespore. The premature prespore-specific σG activity is seen in strains that lack σE activity and is not affected by restoration of σE activity. Ordinarily, σE activity is required both for spoIIIG transcription and for σG activation (14). Thus, σE has lost both roles with respect to σG. In the mutant background, σG becomes active soon after septum formation, as does σF, rather than after completion of engulfment. Although σG is activated earlier, the level of σG activity, as assayed with a PsspA-lacZ transcriptional fusion, is similar to the level that occurs in a spo+ strain. From these results, we suggest that the role of the σE-mediated control is to ensure that activation of σG occurs after the completion of engulfment.

It seems plausible that σE activity is ordinarily needed to reverse some inhibitory signal (or signals) in the prespore, which is not present in the mutant strains. One possibility is that expression of a σF-directed gene in the origin-distal 70% of the chromosome provides an inhibitory signal whose functions include coordinating σG activation with the completion of engulfment. Moving spoIIIG to an origin-proximal site is, in itself, insufficient to overcome this inhibitory signal. However, when the origin-distal region remains trapped in the mother cell in a spoIIIE mutant, there is no inhibitory signal in the prespore and σG becomes active in the absence of σE. With this said, no σF-directed gene encoding such an inhibitory signal has been identified yet. Exclusion of the lonA gene from the prespore in the spoIIIE mutants may also facilitate premature σG activation because σG is particularly sensitive to the LonA protease (36, 39). Another possibility (which does not exclude the possibility described above) is that some structural component of the engulfing septum and/or an origin-distal portion of the chromosome is involved in σG activation. A possible candidate is SpoIIIE. This large, 787-residue protein is already known to have several roles, and it might be part of the mechanism that ensures that σG does not become active until completion of engulfment. It is located in the sporulation septum (51) and is required both for chromosome translocation (50) and for completion of engulfment (41). SpoIIIE forms a single focus in the engulfing septum until engulfment is completed, when it becomes dispersed (41). It may be that changes in SpoIIIE conformation, associated with completion of engulfment, are part of the pathway that relieves σG inhibition. Certainly, loss of SpoIIIE function is pivotal to the system described here for obtaining σE-independent prespore-specific σG activity.

The same changes in genetic background also removed any requirement for SpoIIQ in σG activation. SpoIIQ is made in the prespore. It is normally required for transcription of spoIIIG and some other σF-directed genes, although it is unlikely to act directly on the regulated promoters (48). It may also be required for σG activation and has been shown to interact with SpoIIIAH, whose expression is normally required for σG activation (2). In some conditions it is required for the completion of engulfment (48). The mode of SpoIIQ action remains unclear, although the interaction with SpoIIIAH suggests that SpoIIQ might ordinarily be in the signal pathway from the mother cell that is activated on completion of engulfment. Reinforcing the evidence for such a pathway, the same changes in genetic background that remove any requirement for σE in σG activation also remove the need for SpoIIQ.

Once it becomes active, σG can direct transcription of its own structural gene. This positive feedback loop provides a mechanism for the rapid accumulation of σG when it is needed. However, inappropriate expression can be toxic (21), so the positive feedback loop needs to be tightly controlled. In this report we describe premature activation of σG. Nevertheless, this activation occurs only during spore formation and only in the prespore. It is absolutely dependent on σF activity, and a totally unregulated feedback loop is not established. We previously described a system that leads to mother cell-specific σG activity, which is independent of σF and the anti-sigma factor SpoIIAB (5). Mutations that render σG insensitive to regulation by SpoIIAB also result in inappropriate activation of the feedback loop (40), as do mutations that inactivate lonA, which encodes a protease that can act on σG (36). However, in none of these conditions is σG activity constitutive. Thus, there are several mechanisms to prevent totally unregulated expression of the σG-directed positive feedback loop. The LonA protease is thought to prevent nonspecific activation of σG during stationary phase or during a stress response (36) and may help prevent activation in the mother cell (5). The SpoIIAB protein prevents activation in the mother cell (5, 40). The results reported here support the view that the σE/SpoIIQ system prevents activation of σG in the prespore before completion of engulfment. None of the mechanisms appears to have a role during vegetative growth, suggesting that there may be still other systems regulating σG activity.

Acknowledgments

This work was supported by Public Health Service grant GM43577 to P.J.P. from the National Institutes of Health.

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